<p>The term probiotic is defined as “a live microorganism which, when administered in adequate amounts, confers health benefits on the host.” The genera <i>Lactobacillus</i> and <i>Bifidobacterium</i> are most frequently used as probiotics. Still, probiotic properties have also been reported for other species of genera such as <i>Lactococcus</i>, <i>Enterococcus</i>, <i>Pediococcus</i>, <i>Weissella</i>, <i>Streptococcus</i>, as well as <i>Saccharomyces</i>, <i>Bacillus</i>, and <i>Escherichia coli.</i> Probiotics have significant potential for preventive or therapeutic applications in various diseases such as gastrointestinal disorders, urinary tract infections, allergic diseases, cancer, skin disorders, etc. Therefore, the exact mechanisms of how they work to enhance health and prevent various diseases need to be investigated. Several mechanisms of action have been suggested for probiotics, such as competitive exclusion, production of antimicrobial substances, enhancement of epithelial barrier function, immunomodulation, production of neurotransmitters, antioxidant effects, inhibition of virulence factor expression, and quorum sensing. Understanding these mechanisms provides critical and valuable insights for the development of new-generation probiotics with targeted therapeutic applications. Unlike previous studies that often focus on specific probiotic strains or limited mechanisms, this review provides a comprehensive overview of the multiple functional mechanisms by which probiotics exert their beneficial effects, as well as highlights some of their therapeutic applications. In addition to their mechanisms and beneficial effects, this article also addresses the challenges associated with probiotics, highlighting certain considerations that should be taken into account during their use.</p>